Encapsulated cell indicator system

ABSTRACT

The invention features an encapsulated cell indicator system that includes (a) indicator cells having a signal-responsive element operably linked to a reporter gene; (b) encapsulating material; and (c) a permeable membrane. In this encapsulated cell indicator system, the indicator cells are encapsulated in the encapsulated material and the encapsulated material and the indicator cells are surrounded by the permeable membrane.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority from, U.S.patent application Ser. No. 10/121,295, filed Apr. 12, 2002, whichclaims the benefit of U.S. Provisional Patent Application No. 60/283,838(filed Apr. 13, 2001), each of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

The invention relates to the field of cell transplantation.

The possibility of bone marrow being an in vivo source of circulatingcardiomyocyte progenitors has been suggested. In one experiment,transplanted bone marrow-derived cells were observed to be distributedin a dystrophic mouse heart. Although the molecular characteristics ofthese cells were not identified, their location in the heart tissueindicated these cells were cardiomyocytes. The ability of bone marrowmesenchymal stem cells (BMSCs) to differentiate as beatingcardiomyocytes following introduction of inductive agents such as5-azacytidine has also been shown. Based on these findings, BMSCs havebeen proposed to be a source of cells for treatment of cardiac diseaseand cardiac abnormalities.

Despite the potential therapeutic value of BMSCs, current celltransplantation methods for cardiac tissue are clinically inadequatebecause rate of implant incorporation into the host tissue is poor. Forexample, Orlic et al. (Nature 410: 701-705, 2001) reported that only 40%of mice receiving BMSC transplants showed some myocardial repair.

Thus, there is a need for preparing cells for cell transplantation suchthat there are high rates of cell incorporation and cell survival.

SUMMARY OF THE INVENTION

We have discovered a biologically active indicator system with which todetermine, in real time and in a non-destructive manner, thedifferentiation state of cells in culture. This indicator cell systemallows determination of the differentiation of a first population ofcells being cultured, for example, for transplantation withoutgenetically or otherwise modifying this population to be transplanted.The cell indicator system includes (a) indicator cells having asignal-responsive element operably linked to a reporter gene; (b)encapsulating material; and (c) a permeable membrane. The cell indicatorsystem is co-cultured with the first population of cells. The extent ofreporter gene expression in the indicator system correlates with thedifferentiation state of the second population of cells. Once thereporter gene expression reaches a desired level or the reporter gene isexpressed in a certain percentage of indicator cells, the secondpopulation is collected for use, for example, in cell transplantation.

Accordingly, in a first aspect, the invention features an encapsulatedcell indicator system that includes (a) indicator cells having asignal-responsive element operably linked to a reporter gene; (b)encapsulating material; and (c) a permeable membrane. In thisencapsulated cell indicator system, the indicator cells are encapsulatedin the encapsulating material and the encapsulating material and theindicator cells are surrounded by the permeable membrane.

The indicator cells can include, for example, embryonic stem cells orbone marrow stem cells. While it is desirable that the indicator cellsbe human cells, they can be from any mammal (e.g., a mouse or a pig). Inone embodiment, the cells are the same as the first population of cells,differing in that they include the signal-responsive element and thereporter gene. Any reporter gene can be used in the indicator system, solong as the reporter gene is present in a construct which allows for thereporter gene to be differentially expressed at the stage at which it isdesirable to collect the first population. It is preferable that thedetection of the reporter gene expression can be accomplished by amethod having no substantial toxicity to the cells (e.g., enzymatic andfluorogenic detection methods). Exemplary reporter genes include thoseencoding β-galactosidase, green fluorescent protein, and luciferase.Suitable encapsulating materials include, without limitation, alginate,collagen, gelatin, and chitosan. The encapsulating material can also be,for example, a biodegradable polymer such as polylactic acid (PLA),polyglycolic acid (PGA), or polylactide/glycolide copolymer (PLGA).Exemplary permeable membranes include porous transparent polyethyleneterephthalate (PET) membrane, transparent nylon mesh, transparent porousnylon membrane, and porous transparent polytetrafluoroethylene(PTFE/Teflon).

In a second aspect, the invention features a method of determining thestate of cells in culture without a substantial loss in viability. Thismethod includes (a) providing a culture that includes a population ofcells and the encapsulated cell indicator system of the first aspect,wherein the expression of the reporter gene correlates with the state ofdifferentiation of the population of cells; and (b) measuring theexpression of the reporter gene using a method that does not result insubstantial loss of viability of the population of cells, wherein theexpression level of the reporter gene is indicative of the state ofdifferentiation of the population of cells.

Cells in a particular state of the state of differentiation and suitablegenes from which a signal-responsive element can be derived are listedbelow. Vascular smooth muscle cell: Bves; Endothelial cell: Tie-2, vonWillebrand factor; epicardial cell: Flk-1, ICAM-2; adipocyte: PPAR-γ2;osteoclast: TRAP′ osteoblast: osteocalcin; macrophage: CD11b; neuronalprogenitor: nestin; neuron: neurofilament; astrocyte: GFAP; skeletalmuscle cell: MyoD; smooth muscle cell: SMHC; pancreatic precursor cell:Pdx-1; pancreatic β-cell: hepatocyte: α-fetoprotein.

By “non-destructive,” when referring to an indicator system, is meant amethod that results in less than a 10% loss of cultured cells.Specifically, non-destructive indicators do not require killing thecells of an entire culture vessel in order to determine their state ofdifferentiation.

By “stem cell” is meant a cell capable of (i) self renewing, and (ii)producing multiple differentiated cell types, including one of the groupselected from cardiomyocyte, endothelial cell, and vascular smoothmuscle cell.

By “BMSC” is meant a bone marrow mesenchyme-derived stem cell that isCD45⁻. BMSCs are also referred to as “bone marrow stem cells” and “bonemarrow multipotent progenitor cells.”

As used herein, by “nucleic acid” is meant either DNA or RNA. A “nucleicacid molecule” may be a single-stranded or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases. Unless otherwise specified,the left hand direction of the sequence of a single-stranded nucleicacid molecule is the 5′ end, and the left hand direction ofdouble-stranded nucleic molecule is referred to as the 5′ direction.

By “Csx/Nkx2.5” is meant a nucleic acid or polypeptide that issubstantially identical to the mouse or human Csx/Nkx2.5 cDNA orCsx/Nkx2.5 polypeptide and, when expressed in BMSCs, induces the cellsto become cardiomyogenic. Desirably, the nucleic acid shares at least80% identity with mouse or human Csx/Nkx2.5 over a stretch of 50consecutive nucleotides, more desirably at least 85%, and more desirablyat least 90% or even 95% identity. Gaps of up to 10% may be included inone or both of the sequences. Desirably, the polypeptide shares at least80% identity with mouse or human Csx/Nkx2.5 over a stretch of 25consecutive amino acids, more desirably at least 85%, and more desirablyat least 90% or even 95% identity. Again, gaps of up to 10% may beincluded in one or both of the sequences.

By “treating” is meant reducing or alleviating at least one adverseeffect or symptom of a disorder characterized by insufficient cardiacfunction. Adverse effects or symptoms of cardiac disorders are numerousand well-characterized. Non-limiting examples of adverse effects orsymptoms of cardiac disorders include: dyspnea, chest pain,palpitations, dizziness, syncope, edema, cyanosis, pallor, fatigue, anddeath. For additional examples of adverse effects or symptoms of a widevariety of cardiac disorders, see Robbins, S. L. et al. (1984)Pathological Basis of Disease (W. B. Saunders Company, Philadelphia)547-609; and Schroeder, S. A. et al. eds. (1992) Current MedicalDiagnosis & Treatment (Appleton & Lange, Connecticut) 257-356.

By “disorder characterized by insufficient cardiac function” includes animpairment or absence of a normal cardiac function or presence of anabnormal cardiac function. Abnormal cardiac function can be the resultof disease, injury, and/or aging. As used herein, abnormal cardiacfunction includes morphological and/or functional abnormality of acardiomyocyte or a population of cardiomyocytes. Non-limiting examplesof morphological and functional abnormalities include physicaldeterioration and/or death of cardiomyocytes, abnormal growth patternsof cardiomyocytes, abnormalities in the physical connection betweencardiomyocytes, under- or over-production of a substance or substancesby cardiomyocytes, failure of cardiomyocytes to produce a substance orsubstances which they normally produce, transmission of electricalimpulses in abnormal patterns or at abnormal times, and an alteredchamber pressure resulting from one of the aforementioned abnormalities.Abnormal cardiac function is seen with many disorders including, forexample, ischemic heart disease, e.g., angina pectoris, myocardialinfarction, chronic ischemic heart disease, hypertensive heart disease,pulmonary heart disease (cor pulmonale), valvular heart disease, e.g.,rheumatic fever, mitral valve prolapse, calcification of mitral annulus,carcinoid heart disease, infective endocarditis, congenital heartdisease, myocardial disease, e.g., myocarditis, cardiomyopathy, cardiacdisorders which result in congestive heart failure, and tumors of theheart, e.g., primary sarcomas and secondary tumors.

“Administering,” “introducing,” and “transplanting” are usedinterchangeably and refer to the placement of the cardiomyogenic cellsof the invention into a subject, e.g., a human subject, by a method orroute which results in localization of the cells at a desired site.

By “promoter” is meant a region of nucleic acid, upstream from atranslational start codon, which is involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “humanpromoter” is a promoter capable of initiating transcription in a humancell, and may or may not be derived from a human cell. A “Csx/Nkx2.5promoter” is one derived from the promoter region of a Csx/Nkx2.5 geneand that, when operably linked to a heterologous nucleic acid molecule,is capable of initiating transcription of that molecule (when present ina transcription medium capable of supporting transcription) in a cardiaccell.

By “enhancer element” or “enhancer” is meant a nucleic acid sequencethat, when positioned proximate to a promoter and present in atranscription medium capable of supporting transcription, confersincreased transcription activity relative to the transcription activityresulting from the promoter in the absence of the enhancer domain. A“Csx/Nkx2.5 enhancer” is one derived from the promoter region of aCsx/Nkx2.5 gene and that, when operably linked to a heterologous nucleicacid molecule, is capable of initiating transcription of that molecule(when present in a transcription medium capable of supportingtranscription) in a cardiac cell. A “Tie-2 enhancer” is one derived fromthe promoter region of a Tie-2 gene and that, when operably linked to aheterologous nucleic acid molecule, is capable of initiatingtranscription of that molecule (when present in a transcription mediumcapable of supporting transcription) in an endothelial cell. A “Bvesenhancer” is one derived from the promoter region of a Bves gene andthat, when operably linked to a heterologous nucleic acid molecule, iscapable of initiating transcription of that molecule (when present in atranscription medium capable of supporting transcription) in a vascularsmooth muscle cell.

By “operably linked” is meant that two or more nucleic acid molecules(e.g., a nucleic acid molecule to be transcribed, a promoter, and anenhancer element) are connected in such a way as to permit transcriptionof the nucleic acid molecule in a suitable transcription medium.

By “derived from” is meant that a the nucleic acid molecule was eithermade or designed from a second nucleic acid molecule, the derivativeretaining at least one important-function of the nucleic acid moleculefrom which it was made or designed.

By “expression construct” is meant a nucleic acid molecule that supportstranscription. An expression construct of the present inventionincludes, at the least, a cardiac-specific enhancer element and apromoter. Additional elements, such as a transcription terminationsignal, may also be included, as described herein.

By “vector” or “expression vector” is meant an expression system, anucleic acid-based vehicle, a nucleic acid molecule adapted for nucleicacid delivery, or an autonomous self-replicating circular DNA (e.g., aplasmid). When a vector is maintained in a host cell, the vector caneither be stably replicated by the cells during mitosis as an autonomousstructure, incorporated within the genome of the host cell, ormaintained in the host cell's nucleus or cytoplasm.

By “cardiac cell” is meant a differentiated cardiac cell (e.g., acardiomyocyte) or a cell committed to producing or differentiating as acardiac cell (e.g., a cardiomyoblast or a cardiomyogenic cell).

By “cardiomyocyte” is meant a muscle cell in heart that expressesdetectable amounts of cardiac markers (e.g., alpha-myosin heavy chain,cTnI, MLC2v, alpha-cardiac actin, and, in vivo, Cx43), contracts, anddoes not proliferate.

By “cardiomyoblast” is meant a cell that expresses detectable amountscardiac markers, contracts, and proliferates.

By “cardiomyogenic cell” is meant a cell expressing detectable amountsof Csx/Nkx2.5 RNA or protein, and does not show organized sarcomericstructures or contractions, and preferably does not express detectableamounts of myosin heavy chain protein.

By “epicardial cell” is meant a cell that expresses detectable amountsof Flk-1 and/or ICAM-2, and can become an endothelial cell.

By “endocardial cell” is meant a cardiac cell that expresses detectableamounts of Tie-2 and/or von Willebrand Factor.

By “endothelial cell” is meant a cell that expresses detectable amountsof at least one of the following RNAs or proteins: MUC 18, VE-cadherin,N-cadherin, alpha- and beta-catenins, Flk-1, Tie-2, and CD34.

By “cells primed to differentiate as endothelial cells” is meant stemcells that have not been immortalized that were cultured underconditions that induce the cells to become endothelial cells, wherein atleast about 10%, 25%, 50%, 75%, 90%, 95%, 99%, or even 100% of the cellsare endothelial cells.

By “cells primed to differentiate as vascular smooth muscle cells” ismeant stem cells that have not been immortalized that were culturedunder conditions that induce the cells to become vascular smooth musclecells, wherein at least about 10%, 25%, 50%, 75%, 90%, 95%, 99%, or even100% of the cells are vascular smooth muscle cells.

By “specifically induce one cell type” when referring to differentiationof cultured BMSCs is meant a culture wherein at least 50% of BMSCsdifferentiate into the desired cell type (i.e., cardiomyocytes).

By “detectable amounts” of a protein is meant an amount of a proteinthat is detectable by immunocytochemistry using, for example, themethods provided herein. One method for determining whether a cell isdetectably labeled with either CsX/Nkx2.5 or myosin heavy chain isprovided below. Cultured cells are fixed with 4% formaldehyde for 20minutes on ice, then incubated for 15 minutes in 0.2% Triton X-100 inphosphate-buffered saline (PBS). After three washes in PBS, the cellsare incubated in blotting solution (1% BSA and 0.2% Tween 20 in PBS) for15 minutes. The samples are then treated with one of the followingantibodies: anti-Csx (1:100-1:200, from S. Izumo, Harvard MedicalSchool, Boston Mass.), MF-20 (1:50 to 200, from Developmental StudiesHybridoma Bank, University of Iowa, Iowa City Iowa), anti-desmin(1:100-200, from Sigma-Aldrich, Inc., St. Louis Mo.), and, if desired,their isotype controls (for Csx, normal rabbit serum; for MF-20, mouseIgG2b; for desmin, mouse IgG1) at the same concentration, and incubatedovernight at 4° C. in a moist chamber. The sample slides are then washedthree times using a washing solution (0.5% Tween 20 in PBS) andincubated with secondary antibodies (for Csx, donkey anti-rabbit IgG,for MF-20 and anti-desmin, donkey anti-mouse IgG, all from JacksonImmunoResearch Laboratories, Inc.) following the instructions providedby the vendors, followed by three washes. The samples are then examinedunder a fluorescence microscope (e.g., a Nikon TS100 microscope with amatching fluorescence attachments) and visually scored forimmunolabeling.

By “cardiac-specific enhancer element” is meant an element, operablylinked to a promoter, that directs gene expression in a cardiac cell anddoes not direct gene expression in all tissues or all cell types. Forexample, certain cardiac-specific enhancer elements from Csx/Nkx2.5drive gene expression in cardiac cells as well as in tongue andembryonic stomach. Cardiac-specific enhancers of the present inventionmay be naturally occurring or non-naturally occurring.

By “heterologous” is meant that the nucleic acid molecule originatesfrom a foreign source or, if from the same source, is modified from itsoriginal form. Thus, a “heterologous promoter” is a promoter notnormally associated with the duplicated enhancer domain of the presentinvention. Similarly, a heterologous nucleic acid molecule is modifiedfrom its original form or is from a source different from the sourcefrom which the promoter to which it is operably linked was derived.

By “substantially pure nucleic acid” is meant nucleic acid that is freeof the genes which, in the naturally-occurring genome of the organismfrom which the nucleic acid of the invention is derived, flank thenucleic acid. The term therefore includes, for example, a recombinantnucleic acid which is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic nucleic acid of aprokaryote or a eukaryote cell; or which exists as a separate molecule(e.g., a cDNA or a genomic or cDNA fragment produced by PCR orrestriction endonuclease digestion) independent of other sequences. Italso includes a recombinant nucleic acid that is part of a hybrid geneencoding additional polypeptide sequence.

By “transgene” is meant any piece of a nucleic acid molecule (forexample, DNA) that is inserted by artifice into a cell eithertransiently or permanently, and becomes part of the organism ifintegrated into the genome or maintained extrachromosomally. Such atransgene may include a gene that is partly or entirely heterologous(i.e., foreign) to the transgenic organism, or may represent a genehomologous to an endogenous gene of the organism.

By “transgenic cell” is meant a cell containing a transgene. Forexample, a stem cell transformed with a vector containing an expressionvector operably linked to a heterologous nucleic acid molecule can beused to produce a population of cells having altered phenotypiccharacteristics. A cell derived from a transgenic organism is also atransgenic cell so long as the cells contains the transgene.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a phase contrast micrograph of isolated, cultured bone marrowstem cells from mouse and dog.

FIG. 2 is a series of micrographs showing murine BMSCs following a 14day co-culture with chicken cardiomyocytes.

FIG. 3 is a series of micrographs showing the staining and morphology ofmurine cardiomyocytes, undifferentiated BMSCs, and differentiated BMSCsusing the MF-20 antibody, specific for sarcomeric myosin, or ananti-desmin antibody.

FIG. 4 is a series of micrographs showing the morphology and Csx/Nkx2.5expression of canine BMSCs following in vitro differentiation.

FIG. 5 is a series of micrographs demonstrating in vitro differentiationof murine BMSCs into cells of endothelial lineage.

FIG. 6 is a series of micrographs showing the localization of implantedBMSCs in infarcted dog myocardium 15 days after implantation.

FIG. 7A is a series of micrographs showing the co-localization ofimplanted BMSCs, by DiI fluorescence, and cardiomyocytes, by anti-MHCα/β fluorescence, in the region in which the cells were injected.

FIG. 7B is a micrograph showing increased survival of BMSCs implantedwith a caspase inhibitor, relative to untreated BMSCs.

FIGS. 8A-8D are a series of micrographs showing the co-localization ofimplanted BMSCs, by DiI fluorescence, and cardiomyocytes, by anti-MHCα/β fluorescence in a region away from the site of injection.

FIGS. 9A-9E are a series of micrographs showing the co-localization ofimplanted BMSCs, by DiI fluorescence, and cardiomyocytes, by anti-MHCα/β fluorescence, in a region away from the site of injection.

FIG. 10 is a series of micrographs showing the histopathology of themurine myocardial infarction 36 days after BMSC transplantation.

FIGS. 11-14 are micrographs showing the integration of BMSCs into murinemyocardial tissue, 36 days after transplantation.

FIG. 15 is a pair of micrographs comparing β-galactosidase activity inhCsx-lacZ mouse BMSCs which are cultured in the absence (left panel) orpresence (right panel) of growth factors which induce cardiomyogenicdifferentiation.

FIG. 16A shows an exemplary encapsulated cell indicator system. In theillustration, the indicator cells 2 are encapsulated in an encapsulatingmaterial 4 such as alginate beads. The indicator cells 2 andencapsulating material 4 are contained in a permeable membrane or mesh 6and co-cultured with cells 8 in a culture vessel 10.

FIG. 16B shows the use of encapsulating indicator cells to monitormyogenic differentiation in culture. Shown are micrographs of 11 μm and30 μm nylon mesh, suitable for cell encapsulation; and micrographsshowing the results of the β-galactosidase reaction performed usinghCsx-lacZ mouse BMSC indicator capsules containing varying cell numbers.

FIGS. 17-19 show echocardiograms of infarcted canine heart before (leftpanels) and after (right panels) induced BMSC transplantation.

FIG. 20A is a schematic illustration of an exemplary three-barrel, oneneedle syringe. In this example, each barrel is injected simultaneouslyand evenly into a reservoir adaptor that is connected to a single needlefor a precise injection location. The three syringe barrels areconnected at the top and are controlled by a single plunger depressor.

FIG. 20B is a schematic illustration of a cross-section of thethree-barrel, one needle syringe of FIG. 20A.

FIG. 21A is a schematic illustration of an exemplary three barrel, twoneedle syringe having one larger barrel for injection of one cell type.The two smaller barrels connect to a reservoir adaptor that is connectedto one needle. The larger barrel has a separate needle for injection. Itis desirable that the needle hole of the needle connected to the largerbarrel is increased to maintain the barrel/needle hole ratio of thesmaller barrels, thereby maintaining equal injection pressure in allthree barrels. While optional, the triangular arrangement of the threebarrels allows close proximity of the two needles, while maintaining aparallel injection angle. The three syringe barrels are connected at thetop and are controlled by a single plunger depressor for even injectionpressure.

FIG. 21B is a schematic illustration of a cross-section of thethree-barrel, one needle syringe of FIG. 21A.

FIG. 22A is a schematic illustration of an exemplary three barrel, threeneedle design in which the each syringe barrel has its own needle forinjection. If desired, the triangular arrangement of the three barrelsallows close proximity of the three needles while maintaining a parallelinjection angle. The three syringe barrels are connected at the top andare controlled by a single plunger depressor for even injectionpressure.

FIG. 22B is a schematic illustration of a cross-section of thethree-barrel, one needle syringe of FIG. 22A.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that transplanting developmentally committed butundifferentiated cells will improve the survival, incorporation, andadaptation of the implant in the target tissue.

In developing vertebrates, the early cardiac field is defined by theexpression of the Csx/Nkx2.5 gene. At this developmental stage, however,the Csx/Nkx2.5-expressing cells are still proliferating. We believe thatthe transplantation of Csx/Nkx2.5-expressing cells that are stillproliferating will result in an increased number of incorporated andfunctional cardiomyocytes in the heart.

We have also discovered a therapeutic cellular transplantation method inwhich blood vessels and myocardial tissue are collectively regeneratedin the area of treated myocardium. This method includes thetransplantation of undifferentiated cells committed to become one ofthree cell types: cardiomyocytes, endothelial cells, or vascular smoothmuscle cells.

It is desirable that there be an ample supply of the cells to betransplanted. Accordingly, in one aspect, the cells to be transplantedare derived from stem cells. One suitable stem cell is the BMSC, whichcan be isolated from adult bone marrow. Once isolated, BMSCs can betreated with growth factors (referred to herein as “priming”) to inducethe cells toward a cardiomyocyte cell lineage, as is described below.Alternatively, BMSCs can be primed toward an endothelial cell lineage,or a vascular smooth muscle cell lineage. In one embodiment, the BMSCsare monitored for lineage conversion using a cognate cell type-specificindicator system, such as the one described in U.S. ProvisionalApplication Ser. No. 60/283,837, hereby incorporated by reference. Togenerate the cell type-specific indicator system, transgenic mouse linesare established using a gene construct that includes a celllineage-specific enhancer/promoter-driven marker. For example,cardiomyocyte progenitor conversion can be monitored using encapsulatedBMSCs from hCsx-LacZ transgenic mice. Once adequate marker geneexpression is detected in the cell population, the cells are collectedand injected into the host myocardium.

In one embodiment, the cardiomyocyte progenitor cells, endothelialprogenitor cells, and vascular smooth muscle cells are injectedsimultaneously into the host myocardium. For a proper distribution ofeach cell types in desired areas, a multi-channeled syringe that isdesigned to inject multiple cell types can be used. The length of eachof the needles and the distance between them can be adjusted accordingto the optimal locations of each cell types in the myocardium to berepaired.

Optimization of stem cells and stem cell derivative preparations iscritical for successful cell transplantation. To achieve maximum yieldin cell transplantation, the implanted cells are desirably at the properstage of commitment and differentiation. Currently, despite the knowncommitment and differentiation markers for many animal cells, it isdifficult to determine the proper time to harvest cells during in vitroculture without performing time-consuming molecular biological assaysfor the expression of these markers. We have discovered a biologicallyactive indicator system with which to determine, in real time, thedifferentiation state of cells in culture. This indicator system is alsouseful, for example, for determining the amount of gene expression ofproteins during cell growth or cell death.

Most tissue-specific gene expression is controlled by enhancer andrepressor sequences at the transcriptional level. Generally, to confertightly-regulated expression, enhancers adopt complex regulatorymechanisms that require the collaboration of multiple transcriptionfactors. The binding sites for these transcription factors may be manykilobases (kb) from the gene promoter and dispersed relative to eachother.

When used to drive transgene expression in mice, cardiac enhancers fromhCsx/Nkx2.5 and mCsx/Nkx2.5 recapitulate expression patterns of theendogenous mCsx/Nkx2.5 (see, e.g., U.S. Patent Application PublicationNo. 2002022259, hereby incorporated by reference). Among the mammaliancardiac enhancers known so far, one of these enhancers (the 7.5 kbenhancer) is the earliest enhancer that is active in all four heartchambers. Moreover, this enhancer displays no ectopic expression. Withinthis 7.5 kb fragment, two regions (referred to herein as homology domainA1 and homology domain A2 were isolated that together, when operablylinked to an hsp68 promoter-lacZ cassette, were capable of enhancinggene expression in a cardiac-specific manner. These two regions can alsobe used in the reporter constructs of the invention.

EXAMPLE 1 Induction of Cardiomyogenic Cells from BMSCs

Marrow was isolated from adult mouse and dog. The BMSCs were isolatedand cultured in medium containing 10% fetal bovine serum, 100 μML-ascorbic acid-2-PO₄, 5-15 ng/ml leukemia inhibitory factor (LIF), and20 nM dexamethasone (for mouse cultures, mouse LIF was used, while fordog cultures, human LIF was used). This in vitro condition allows theBMSCs to maintain their self-renewing character and to expand bypassaging without losing responsiveness to the differentiation agentssuch as growth factors. Further, stem cells cultured through multiplepassages maintain a mesenchymal morphology and karyotype (FIG. 1). After14 days in culture with growth factors (50 ng/ml BMP2, 100 ng/ml bFGF),approximately 80% of the BMSCs were positively stained with Csx/Nkx2.5,MF-20 (a monoclonal antibody specific for sarcomeric myosin), and desminantibodies, indicating that the cells had differentiated ascardiomyocytes (FIGS. 3 and 4).

To mimic the environment of the adult myocardium to which transplantedBMSCs are exposed, a co-culture model system was used. In this system,BMSCs, labeled with a fluorescent tag for identification (Vybrant™), andprimary chicken embryonic cardiomyocytes were co-cultured, at a ratio of1:40, on glass slides coated with 5 ng/ml collagen. These mixed cultureswere grown alone, in the presence of 25 ng/ml BMP2, or in the presenceof 25 ng/ml bFGF. Cells were subsequently stained with anti-Csx/Nkx2.5,MF-20 (a monoclonal antibody specific for sarcomeric myosin), andanti-desmin antibodies. Five days after the initiation, a few (0.1-1%)myosin-positive cells were detected in co-cultures grown in the presenceof BMP2 or bFGF, while co-cultures grown in the absence of either growthfactor were negative for all three antibodies. However, after two weeksof co-culture in the absence of growth factors, numerous BMSCs wereMF-20-positive, suggesting they had converted to a myogenic cell lineage(FIG. 2). Thus, BMSCs can be induced to differentiate along a myogeniclineage using either growth factors such as BMP2 and bFGF, or byco-culture with differentiated cardiomyocytes.

In view of the foregoing results, we can regulate the rate and amountthat BMSCs become cardiomyogenic cells in culture by modulating theenvironment in which the cells are cultured. According to thetransplantation method of the invention, it is desirable that at least10% of the transplanted cells be cardiomyogenic cells (i.e., mitoticcells that express Csx/Nkx2.5 but do not show organized sarcomericstructures or contractions, and preferably do not express detectableamounts of myosin heavy chain RNA or protein). A higher percentagecardiomyogenic cells will result in increased incorporation of implantedcells. Thus, is it desirable that at least 10%, 25%, 50%, 75%, 85%, 90%,or 95% or more of the cells be cardiomyogenic cells. Real-timemeasurement of commitment can be performed using the cell indicatorsystem described in Example 5, below.

EXAMPLE 2 BMSCs from Humans and Other Mammals

The foregoing example utilizes mouse BMSCs for illustrative purposes.Human BMSCs are also known in the art to be capable of producing cardiaccells (Pittenger et al., Science 284: 143-147, 1999). BMSCs from othermammals (e.g., humanized pig BMSCs) can also be used in the methods ofthe invention (Levy et al., Transplantation 69: 272-280, 2000).

EXAMPLE 3 Methods of Inducing BMSCs to Become Cardiomyogenic

As is described above, co-culturing BMSCs with cardiomyocytes in thepresence of BMP2 and/or bFGF results in the induction of cardiomyogeniccells capable of differentiating as cardiomyocytes in culture. The ratioof BMSCs to inducer cells and the concentration of growth factor(s) caneach be adjusted to modulate the rate and amount of cardiomyogenic cellinduction. For example, the ratio of BMSCs to inducer cells can rangefrom about 1:1 to about 1:1000 or more. The concentration of BMP2 canrange from about 0.5 ng/ml to about 1 μg/ml, while the concentration ofbFGF can range from about 1 ng/ml to about 5 μg/ml. It is understoodthat other BMP/TGFβ and FGF family members can be used instead of BMP2and/or bFGF.

Other methods known to induce BMSCs to become cardiomyogenic cells canbe used in the present invention. Not all methods that inducecardiomyocytes can be used in the invention. For example, 5-azacytidineis used as the inducing agent for cardiomyocytes (Makino et al., J.Clin. Invest., 103: 697-705, 1999) but is not appropriate in the methodsof the invention. Since 5-azacytidine randomly demethylates genomicsequences (thereby inducing normally silent genes), treatment of theBMSCs with 5-azacytidine can generate a variety of cell types (e.g.,myocytes (MyoD positive), osteoblasts (osteocalcin positive), andadipocytes (PPAR-γ positive)), in addition to cardiomyocytes (cardiactroponin I positive) (Wakitani et al., Muscle Nerve, 18: 1417-1426,1995; Tomita et al., Circulation, 100 suppl II: 247-256, 1999). BMSCsexposed to 5-azacytidine are known to rapidly upregulate c-abl andinterleukin-6 transcripts while downregulating the expression ofcollagen I, a major matrix protein. (Andrews et. al., Mol. Cell. Biol.,9: 2748-2751, 1989). In the methods of the invention, suitable factorsor conditions are those that specifically induce one cell type (e.g.,cardiomyocytes).

EXAMPLE 4 Induction of Other Cell Types

It may be desirable to induce cells types such as vascular smooth musclecells and endothelial cells (or their precursors) for transplantationinto the myocardium because theses cells may generate new blood vesselsaround the transplanted cardiomyogenic cells. The cells can betransplanted alone, but preferably are transplanted with the appropriatecardiomyogenic cells, as described herein.

Differentiation of vascular smooth muscle cells can be determined usingthe Bves gene enhancer (Reese et al., Dev. Biol. 209: 159-171, 1999).Differentiation of endothelial cells can be determined using Tie-2 orvon Willebrand Factor enhancers that have been cloned (Schnurch andRisau, Development 119: 957-968, 1993 and Coffin et. al., Dev. Biol.148: 51-62, 1991, respectively). Differentiation of embryonic epicardialcells (i.e., precursors of endothelial cells) can be determined usingFlk-1 or ICAM-2 enhancers (Shalaby et al., Nature 376:62-66, 1995 andTevosian et al., Cell 101:729-739, 2000, respectively).

BMSCs were isolated as describe above and cultured in the presence ofbiological factors known to generate endothelial cell lineages duringembryonic development (2% FBS, 20 ng/ml VEGF, 1 ng/ml bFGF, and 2 ng/mlIGF-I). Flk-1, an endothelial-specific receptor tyrosine kinase, wasrobustly expressed in approximately 80% of cultured BMSCs, after 14 daysin culture, indicating conversion to an endothelial cell lineage (FIG.5).

EXAMPLE 5 Cell Indicator System

As depicted in FIG. 16A, the indicator system includes three components:indicator cells 2, a cell encapsulation system (CES) 4, and a permeableoutside membrane or mesh 6 that helps retain the indicator cells in theCES and separates the indicator cells 2 from those to be transplanted 8.

In one example, the indicator system is useful in determining the stateof cell commitment and differentiation of stem cells (e.g., BMSCs). Asis described herein, it is desirable to prime human BMSCs such thatabout 5-100% (preferably about 80%) of the cells are cardiomyogeniccells (as determined by Csx/12.5 expression, the lack of organizedsarcomeric structures or contractions, and preferably, the lack ofmyosin heavy chain RNA or protein). Thus, an extremely rapid assay isdesired in order to minimize the time interval between harvesting thecells for the assay and transplantation. A rapid assay, therefore,ensures that the assay results are representative of theCsx/Nkx2.5-expressing cells which are ultimately transplanted. Thepresent invention provides such an assay. BMSCs from transgenic micecontaining a Csx enhancer operably linked to a reporter gene are used asindicator cells. Suitable Csx enhancers are described, for example, inU.S. Patent Application Publication No. 2002022259, hereby incorporatedby reference. The indicator cells are either encapsulated in abiological material (e.g., alginate, collagen, gelatin, or chitosan) orattached onto a biodegradable polymer (e.g., non-porous microspheres ofpolylactic acid (PLA), polyglycolic acid (PGA), or polylactide/glycolidecopolymer (PLGA)). The encapsulated or microsphere-attached cells arethen surrounded by a membrane that permeable to oxygen, nutrients, andother biomolecules. Examples of suitable membranes include poroustransparent polyethylene terephthalate (PET) membrane, transparent nylonmesh, transparent porous nylon membrane, and porous transparentpolytetrafluoroethylene (PTFE/Teflon).

In addition to retaining the indicator cells in the capsule, the outermembrane provides a physical integrity to the system. During theinduction of the human BMSCs into cardiomyogenic cells, the reportergene operably linked to the Csx enhancer (e.g., a human Csx enhancer)will be expressed in the indicator cells. Nontoxic detection of reportergene expression indicates the differentiation state of the human cells.Suitable reporter genes include, without limitation, those encodinggreen fluorescent protein, β-galactosidase, and luciferase. Afterdetermining that the cells have reached the desired state ofdifferentiation, the entire indicator system (including the indicatorcells, the encapsulating material, and permeable membrane) is removed.The cells to be implanted are then collected and prepared fortransplantation. If desired, the cells can be frozen and stored untiltransplantation.

In the cell indicator system of the present invention, the indicatorcells can be any cell type in which the enhancer element/reporter geneconstruct is operable as the cells differentiate. In one example, BMSCcells transfected with the reporter construct are used. These cells canbe any animal BMSCs or, alternatively, other cell types such as ES cellstransfected with enhancer element/reporter gene construct, or BMSCs froman enhancer element/reporter gene transgenic animals.

We demonstrate the principles described above, of an encapsulated cellindicator system, using murine BMSCs derived from hCsx-lacZ transgenicmice. We have found that BMSCs which are not induced to differentiatealong a cardiomyogenic lineage stain weakly or not at all following astandard β-galactosidase assay. (FIG. 15). In contrast, BMSCs culturedaccording to the methods described above, which induce cardiomyogenicdifferentiation, produce a strongly positive signal (FIG. 15).Accordingly, the murine hCsx-lacZ BMSCs are excellent candidates forencapsulation as a system that can be used to monitor the progression ofmyogenic differentiation (FIGS. 16A and 16B). Such an indicator systemprovides several advantages over traditional techniques for assessingcell differentiation. Specifically, the capsules are easily recoveredfrom the culture media and can be rapidly and reliably assayed. Further,because the capsule can be incorporated and recovered from every culturevessel, monitoring can be done on a plate-by-plate basis. It is notnecessary to destroy an entire culture for monitoring purposes, as isrequired using traditional histological techniques. This is particularlyimportant when using BMSCs from a human patient where bone marrowsamples are difficult to obtain and few stem cells are available forculture and transplantation.

Murine hCsx-lacZ BMSCs can be encapsulated in any appropriate materialwhose properties are described above. Useful capsules can be made, forexample, by embedding the cells in alginate and containing thealginate-embedded cells in 11 μm or 30 μm nylon mesh, available, forexample, from Millipore Corp. (Bedford, Mass.), which is both durableand permeable to culture media and growth factors, oxygen, and chemicalreagents used in the β-galactosidase assay. Using the methods describedherein, capsules are desirably formed in solutions containing at leastabout 10⁶ hCsx-lacZ BMSCs per milliliter; however the use of at leastabout 10⁷ cells/ml is more desirable. Of course, as conditions vary, aperson of ordinary skill could determine the appropriate concentrationof indicator cells in the capsule system.

The specific indicator cells used to create the encapsulated monitoringsystem on this invention need not be murine cells. The indicator cellscan be either heterologous or autologous to the transplant recipient. Incases where BMSCs are relatively plentiful, it is preferable totransfect a subset of the host BMSCs with a reporter construct, such asthe one previously described. These autologous BMSCs are thenencapsulated and used for monitoring purposes. Alternatively, in caseswhere BMSCs are in limited supply, non-autologous (homologous orheterologous) indicator BMSCs can be used.

EXAMPLE 6 Methods for Transplantation

The invention pertains to methods for treating disorders characterizedby insufficient cardiac function in a subject by autologous orheterologous cardiac cell transplantation. The methods includeadministering to the subject the stem cell-derived cardiomyocyteprogenitors, endothelial cell progenitors, and vascular smooth muscleprogenitors of the invention, which are described in detail herein.Transplantation of the cells of the invention into the heart of thesubject with a cardiac disorder results in replacement of lost ornon-functioning (“hybernating”) cardiomyocytes. The cells are introducedinto a subject with a cardiac disorder in an amount suitable to replacelost or non-functioning cardiomyocytes such that there is an at leastpartial reduction or alleviation of at least one adverse effect orsymptom of the cardiac disorder. The cells can be administered to asubject by any appropriate route that results in delivery of the cellsto a desired location in the subject where at least a portion of thecells remain viable. It is desirable that at least about 5%, desirablyat least about 10%, more desirably at least about 20%, yet moredesirably at least about 30%, still more desirably at least about 40%,and most desirably at least about 50% or more of the cells remain viableafter administration into a subject. The period of viability of thecells after administration to a subject can be as short as a few hours,e.g., twenty-four hours, to a few days, to as long as a few weeks tomonths. One method that can be used to deliver the cells of theinvention to a subject is direct injection of the cells into theventricular myocardium of the subject (e.g., Soonpaa et al., Science264:98-101, 1994; Koh et al., Am. J. Physiol. 33:H1727-1733, 1993). Thecells can be administered in a physiologically compatible carrier, suchas a buffered saline solution. To treat disorders characterized byinsufficient cardiac function in a human subject, about 10⁴-10⁹ cellsare introduced into the human, e.g., into the myocardium.

To accomplish these methods of administration, the cells of theinvention can be inserted into a delivery device that facilitatesintroduction by injection or implantation of the cells into the subject.Such delivery devices include tubes, e.g., catheters, for injectingcells and fluids into the body of a recipient subject. In a preferredembodiment, the tubes additionally have a needle or needles throughwhich the cells of the invention can be introduced into the subject at adesired location. It may be desirable to maintain each cell type in adifferent set of conditions (such as in different media) during theinjection. In such a case, a multi-barrel syringe with one, two, orthree needles can be used for injection (FIGS. 20A, 20B, 21A, 21B, 22A,and 22B). If a three-barrel/two-needle syringe is used, it is preferablethat endothelial cell progenitors and smooth muscle cell progenitors bemixed during the injection.

The cells of the invention can be inserted into such a delivery devicein different forms. For example, the cells can be suspended in asolution or embedded in a support matrix when contained in such adelivery device. Preferably, the solution includes a pharmaceuticallyacceptable carrier or diluent in which the cells of the invention remainviable. Pharmaceutically acceptable carriers and diluents includesaline, aqueous buffer solutions, solvents and/or dispersion media. Theuse of such carriers and diluents is well known in the art. The solutionis preferably sterile and fluid. Preferably, the solution is stableunder the conditions of manufacture and storage and preserved againstthe contaminating action of microorganisms such as bacteria and fungithrough the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, or thimerosal. Solutions of the invention can be preparedby incorporating the cells as described herein in a pharmaceuticallyacceptable carrier or diluent and, as required, other ingredients.

Support matrices in which the cells of the invention can be incorporatedor embedded include matrices which are recipient-compatible and whichdegrade into products that are not harmful to the recipient. Naturaland/or synthetic biodegradable matrices are examples of such matrices.Natural biodegradable matrices include, for example, collagen matricesand alginate beads. Synthetic biodegradable matrices include syntheticpolymers such as polyanhydrides, polyorthoesters, and polylactic acid.These matrices provide support and protection for the cells in vivo.

Prior to introduction into a subject, the cells can be modified toinhibit immunological rejection. For example, to inhibit rejection oftransplanted cells and to achieve immunological non-responsiveness in atransplant recipient, the method of the invention can include alterationof immunogenic antigens on the surface of the cells prior tointroduction into the subject. This step of altering one or moreimmunogenic antigens on the cells can be performed alone or incombination with administering to the subject an agent that inhibits Tcell activity in the subject. Alternatively, inhibition of rejection ofthe transplanted cells can be accomplished by administering to thesubject an agent that inhibits T cell activity in the subject in theabsence of prior alteration of an immunogenic antigen on the surface ofthe transplanted cells. An agent that inhibits T cell activity isdefined as an agent which results in removal (e.g., sequestration) ordestruction of T cells within a subject or inhibits T cell functionswithin the subject. T cells may still be present in the subject but arein a non-functional state, such that they are unable to proliferate orelicit or perform effector functions (e.g., cytokine production,cytotoxicity, etc). The agent that inhibits T cell activity may alsoinhibit the activity or maturation of immature T cells (e.g.,thymocytes). A preferred agent for use in inhibiting T cell activity ina recipient subject is an immunosuppressive drug that inhibits orinterferes with normal immune function. A preferred immunosuppressivedrug is cyclosporin A. Other immunosuppressive drugs that can be usedinclude, for example, FK506 and RS-61443. In one embodiment, theimmunosuppressive drug is administered in conjunction with at least oneother therapeutic agent. Additional therapeutic agents that can beadministered include steroids (e.g., glucocorticoids such as prednisone,methyl prednisolone, and dexamethasone) and chemotherapeutic agents(e.g., azathioprine and cyclosphosphamide). In another embodiment, animmunosuppressive drug is administered in conjunction with both asteroid and a chemotherapeutic agent. Suitable immunosuppressive drugsare commercially available.

In addition to its use in the treatment of cardiac-related disorders,cell transplantation therapy is applicable to a wide variety of diseasesand disorders (e.g., Parkinson's disease, diabetes, spinal cord injury,multiple sclerosis). As with transplantation into the myocardium, thetransplantation of mitotic cells that are competent and primed to adoptthe desired cell fate will likely aid in the integration of thetransplanted cells, resulting in more of the desired cells incorporatingand surviving in the host tissue. Enhancers useful for the detection ofthe differentiation, commitment, or competence of a cell lineage aredepicted in Table 1, below.

TABLE 1 Cell Type Marker Reference Vascular smooth muscle Bves Reese etal., Dev. Biol. 209: 159-171, 1999. cell Endothelial cell Tie-2 Schnurchand Risau, Development 119: 957-968, 1993. von Willebrand Coffin et al.,Dev Biol. 148: 51-62, 1991. Epicardial cell Flk-1 Shalaby et al., Nature376: 62-66, 1995. ICAM-2 Tevosian et al., Cell 101: 729-39, 2000.Adipocyte PPAR-g2 Zhu et al., PNAS 92: 7921-7925, 1995. Osteoclast TRAPReddy, J. Bone Miner. Res. 10: 601-606, 1995. Osteoblast OsteocalcinKesterson, Mol Endocrinol. 7: 462-467, 1993. Macrophage CD11b Dzienniset al., Blood. 85: 319-329, 1995. Neuronal progenitor Nestin Yamaguchiet al., Neuroreport 11: 1991-1996, 2000. Neuron Neurofilament Leconte etal., J Mol Neurosci 5: 273-295, 1994. Astrocyte GFAP Nolte et al., Glia.33: 72-86, 2001. Skeletal muscle cell MyoD Goldhammer et al., Science256: 538-42, 1992. Smooth muscle cell SMHC Zilberman et al., Circ Res1998 82: 566-575, 1998. Pancreatic precursor cell Pdx-1 Marshak et al.,Mol. Cell Biol. 20: 7583-7590, 2000. Pancreatic β-cell GlucokinaseJetton, et al., JBC. 269: 3641-3654, 1993. Hepatocyte α-fetoproteinGhebranious, Dev 42: 1-6, 1995. Each of the foregoing references ishereby incorporated by reference.

EXAMPLE 7 Canine Model of Myocardial Infarction

BMSCs, which were directed toward a cardiogenic cell lineage in vitro,were transplanted into infarcted dog myocardial tissue. The dogmyocardial infarction was created by permanent occlusion of the leftcoronary artery. The infarction was allowed to stabilize for at leasttwo months prior to BMSC transplantation. In order to preventimmunorejection of the transplants, marrow was collected and BMSCsprepared from the individual transplant recipient dogs as follows. Aboutfour weeks after the ligation, after the myocardial infarction had beenconfirmed using echocardiogram, iliac bone was punctured to aspiratebone marrow. Bone marrow was immediately mixed with heparin, frozen andtransported in dry ice to the tissue culture facility, where the bonemarrow was thawed at 37° C., perturbated, washed once with regular DMEM,and plated in tissue culture flasks containing culture medium (10% fetalbovine serum, 100 μM L-ascorbic acid-2-PO₄, 5-15 ng/ml LIF, and 20 nMdexamethasone). At the time of harvest, BMSCs were labeled with DiI, ared fluorescent marker, to track the survival and progression of thecells following transplantation. The labeled BMSCs were then cultured inthe presence of 100 ng/ml bFGF for 4-7 days. Cells (1.5-250 million)were harvested and injected into the infarcted region of the heart.

BMSC survival following transplantation was assessed by post-mortemvisualization of DiI fluorescence. Large clusters of DiI-positive cellswere observed in the myocardium 15 days after transplantation,suggesting long-term viability of the BMSCs (FIG. 6). Specifically, theDiI-labeled stem cells were observed within regions of the myocardiumcontaining MF-20-positive cardiomyocytes and in the infarcted regionswhich were devoid of MF-20-positive cardiomyocytes (FIG. 6). Further,the border region of the infarcted area contained DiI positive stemcells which also express the cardiac muscle-specific marker MHC a/p(FIGS. 7-9). Together, these data demonstrate that transplanted BMSCs,which have been conditioned in vitro according to the described methods,survive and incorporate into the host myocardium and express markerscharacteristic of cardiac differentiation.

EXAMPLE 8 BMSC Implantation Reduces Infarction Size

The canine myocardial infarction model described in Example 7 was usedfor in vivo assessment, by echocardiogram (ECG), of the restorativeeffects of BMSC transplantation. ECGs were performed 3.5, 4.5, and 5weeks after BMSC transplantation (FIGS. 19, 17, and 18, respectively)and compared to pre-implantation ECGs. In each animal, contraction ofthe infarct area became more synchronized with neighboring area of themyocardium. Thus, the ECG results confirm the histological findings ofExample 7 and demonstrate that transplantation of stimulated, culturedBMSCs results in a partial restoration of cardiac tissue followinginfarction.

EXAMPLE 9 Murine Model of Myocardial Infarction

The long-term viability of implanted BMSCs was also investigated using amurine cardiac infarction model system. In order to preventimmunorejection, BMSCs were isolated from marrow collected from miceisogenic to those used for transplantation. As described above, theBMSCs were cultured in the presence of 100 ng/ml bFGF for 4-7 days, thenfluorescently labeled with DiI and harvested. Infarctions were createdby left coronary artery banding. The treated BMSCs were then injectedinto the infarction area as follows. BMSCs (100,000 to 500,000 in 10 μlPBS or HBSS) were injected in the anteroseptal LV myocardium in anoblique way, using a 50 microliter Hamilton syringe with a matching 29 Gor 30 G Hamilton needle. During the surgery, the mouse was kept on acustom made heated bed maintained at 37° C. using a feedback temperaturecontroller, and respiration was assisted using a mouse respirator (setvolume 200 microliters, 110/min rate). Thirty-six days aftertransplantation, the infarcted area was analyzed for the presence ofDiI-labeled cells and cardiomyocyte viability. As observed in the caninemodel, labeled BMSCs were incorporated within the murine myocardialinfarct. Further, DiI-labeled cells present in the myocardium exhibitedmorphologies characteristic of cardiomyocytes. Hematoxylin and eosinstaining of this region shows striations, corkscrew nuclei, andelongated fibers that are characteristic of cardiac muscle (FIG. 10)inside of the infarct. We also observed DiI-positive cells adjacent tothe infracted area of the myocardium. Visualization of cardiomyocytesusing α-MHC, MF-20, and cardiac troponin antibody staining demonstratedthat the DiI-labeled cells (transplanted BMSCs) were located completelywithin the cardiac myofibrils (FIGS. 11, 12, and 14). Transplanted BMSCswere also incorporated in the neighboring regions of the myocardium(FIG. 13). Thus, the transplanted stimulated BMSCs fully integrated intoboth the normal and infarcted cardiac tissue and continueddifferentiating into cardiomyocytes; a process begun prior totransplantation, during the in vitro stimulation.

EXAMPLE 10 Methods of Inducing Stem Cells to Become EndothelialProgenitor Cells

To generate primed endothelial progenitor cells, isolated stem cells(e.g., human BMSCs) are primed using VEGF (10 ng/ml), bFGF (1 ng/ml),and IGF-I (2 ng/ml) for a period of 4-7 days (Shi et al., Blood92:362-367, 1998). As a conversion indicator in the cell indicatorsystem, stem cells containing a Tie enhancer operably linked to areporter gene can be used (Schlaeger et al., Proc. Natl. Acad. Sci. USA94:3058-3063, 1997).

EXAMPLE 11 Methods of Inducing Stem Cells to Become Vascular SmoothMuscle Progenitor Cells

To generate primed vascular smooth muscle progenitor cells, isolatedstem cells (e.g., human BMSCs) can be primed using PDGF (1-10 ng/ml) andTGF-β (1-10 ng-ml) for a period of 4-7 days (Hirschi et al., J. CellBiol. 141:805-814, 2000). As a conversion indicator in the cellindicator system, stem cells containing a Bves enhancer operably linkedto a reporter gene can be used (Reese et al, Dev. Biol. 209:159-171,1999).

Other Embodiments

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. An encapsulated cell indicator system comprising: (a) indicator cellscomprising a signal-responsive element operably linked to a reportergene; (b) encapsulating material; and (c) a permeable membrane, whereinsaid indicator cells are encapsulated in said encapsulating material andsaid encapsulating material and said indicator cells are containedwithin said permeable membrane.
 2. The encapsulated cell indicatorsystem of claim 1, wherein said indicator cells comprise embryonic stemcells or bone marrow stem cells.
 3. The encapsulated cell indicatorsystem of claim 1, wherein said indicator cells comprise human cells. 4.The encapsulated cell indicator system of claim 1, wherein saidindicator cells comprise mouse cells or pig cells.
 5. The encapsulatedcell indicator system of claim 1, wherein said reporter gene encodesβ-galactosidase, green fluorescent protein, or luciferase.
 6. Theencapsulated cell indicator system of claim 1, wherein saidencapsulating material comprises alginate, collagen, gelatin, chitosan,polylactic acid, polyglycolic acid, or polylactide/glycolide copolymer.7. The encapsulated cell indicator system of claim 1, wherein saidpermeable membrane comprises polyethylene terephthalate membrane, nylonmesh, porous nylon membrane, or porous polytetrafluoroethylene(PTFE/Teflon).
 8. A method of determining the differentiation state ofcells in culture, said method comprising the steps of: (a) providing apopulation of cells and an encapsulated cell indicator system, saidencapsulated cell indicator system comprising: (i) indicator cellscomprising a signal-responsive element operably linked to a reportergene; (ii) encapsulating material; and (iii) a permeable membrane,wherein said indicator cells are encapsulated in said encapsulatingmaterial and said encapsulating material and said indicator cells arecontained within said permeable membrane, wherein the expression of saidreporter gene correlates with the differentiation state of saidpopulation of cells; and (b) measuring the expression of said reportergene while said cells are contained within said permeable membrane usinga method that does not result in substantial loss of viability of saidpopulation of cells, wherein said expression level of said reporter geneis indicative of the state of differentiation of said population ofcells.
 9. The method of claim 8, wherein said indicator cells compriseembryonic stem cells or bone marrow stem cells.
 10. The method of claim8, wherein said indicator cells comprise human cells.
 11. The method ofclaim 8, wherein said indicator cells comprise mouse cells or pig cells.12. The method of claim 8, wherein said reporter gene encodesβ-galactosidase, green fluorescent protein, or luciferase.
 13. Themethod of claim 8, wherein said encapsulating material comprisesalginate, collagen, gelatin, chitosan, polylactic acid, polyglycolicacid, or polylactide/glycolide copolymer.
 14. The method of claim 8,wherein said permeable membrane comprises polyethylene terephthalatemembrane, nylon mesh, porous nylon membrane, or porouspolytetrafluoroethylene (PTFE/Teflon).
 15. A method of preparing bonemarrow stem cells for transplantation into cardiac tissue of a patient,said method comprising the steps of: (a) providing a population of bonemarrow stem cells from said patient and an encapsulated cell indicatorsystem, said encapsulated cell indicator system comprising: (i)indicator cells comprising a signal-responsive element operably linkedto a reporter gene; (ii) encapsulating material; and (iii) a permeablemembrane, wherein said indicator cells are encapsulated in saidencapsulating material and said encapsulating material and saidindicator cells are contained within said permeable membrane, whereinthe expression of said reporter gene correlates with the differentiationstate of said bone marrow stem cells; (b) culturing said population ofbone marrow stem cells and said encapsulated cell indicator system underconditions that induce said bone marrow stem cells to differentiate ascardiac cells; (c) measuring the expression of said reporter gene whilesaid cells are contained within said permeable membrane using a methodthat does not result in substantial loss of viability of said populationof cells; and (d) collecting said bone marrow stem cells when theexpression of said reporter gene indicates that at least some of saidbone marrow stem cells have been induced to differentiate as cardiaccells.
 16. The method of claim 15, wherein said permeable membrane istransparent or semitransparent.